Process for the preparation of nanostructures on a dental implant

Abstract

A process for the preparation of a topography for improved fibrin network formation and cell mineralization on at least a portion of a dental implant made of a binary titanium-zirconium alloy, the portion being destined to be embedded in a patient's jawbone and to be in contact with the jawbone via a bone-contacting surface, the process includes the subsequent steps of a) subjecting the bone-contacting surface of the dental implant to a sandblasting treatment, b) etching the sandblasted bone-contacting surface, and c) treating the sandblasted and etched bone-contacting surface with water or an aqueous solution for a duration of more than two days, during which nanostructures continuously grow on the bone-contacting surface, the nanostructures extending in at least two dimensions to 200 nm at most. The process is characterized in that the treatment of b) is carried out at a temperature from 40° C. to 60° C.

Claims

1. A process for the preparation of a topography for improved fibrin network formation and cell mineralization on at least a portion of a dental implant made of a binary titanium-zirconium alloy, the portion being configured to be embedded in a jawbone of a patient and to be in contact with the jawbone via a bone-contacting surface, the process comprising a) subjecting the bone-contacting surface of the dental implant to a sandblasting treatment, b) etching the sandblasted bone-contacting surface, and c) treating the sandblasted and etched bone-contacting surface with water or an aqueous solution for a duration of more than two days and no more than 4 weeks, during which nanostructures continuously grow on the bone-contacting surface, the nanostructures extending in at least two dimensions to 200 nm at most, wherein: the treatment of step c) is carried out at a temperature in a range from 40° C. to 60° C., prior to step c), the dental implant is packed in a dental implant packaging, in which the dental implant is immersed in the water or the aqueous solution, and the packaging is made of a material selected from the group consisting of cyclic olefin copolymer (COC), polyethylene (PE), polypropylene (PP), polyether ether ketone (PEEK), polyether ketone ketone (PEKK), and polytetrafluoroethylene.

2. The process according to claim 1, wherein the treatment of step c) is carried out for a duration of 5 days at least.

3. The process according to claim 1, wherein the treatment of step c) is carried out at a temperature in a range from 50° C. to 60° C.

4. The process according to claim 1, wherein after the treatment of step c), the dental implant is stored in the dental implant packaging that is sealed until use, whereby during the storing, the dental implant is kept immersed in the water or the aqueous solution used for the treatment of step c).

5. The process according to claim 1, wherein the packaging comprises at least two packaging components of different polymeric materials.

6. The process according to claim 1, wherein the aqueous solution has a pH value ranging from 2 to 10.

7. The process according to claim 1, wherein the aqueous solution is a physiologic salt solution.

8. The process according to claim 1, wherein the dental implant is subjected to a sterilization treatment prior to step c).

9. The process according to claim 1, wherein for the etching according to step b), an etching solution comprising a mineral acid is used.

10. The process according to claim 9, wherein the etching solution comprises a mixture of HCl and H.sub.2SO.sub.4.

11. The process according to claim 1, wherein the dental implant is made of a binary titanium-zirconium alloy containing from 13 to 17 wt-% of zirconium.

12. A dental implant obtainable by the process according to claim 1, the implant being made of the binary titanium-zirconium alloy and comprising the portion that is configured to be embedded in the jawbone of the patient and to be in contact with the jawbone via the bone-contacting surface on which the nanostructures extending in at least two dimensions to 200 nm at most have been grown.

13. The dental implant according to claim 12, wherein the nanostructures have a length-to-diameter ratio of more than 1 to 1.

14. The dental implant according to claim 12, wherein the bone-contacting surface has a hydrophilicity defined by a contact angle of less than 45° when contacted with water.

Description

(1) The results are shown in the attached Figures of which

(2) FIG. 1 shows an SEM picture of the sample obtained by the process of the present invention after a treatment of 1 week;

(3) FIG. 2 shows an SEM picture of the sample obtained by the process of the present invention after a treatment of 2 weeks;

(4) FIG. 3 shows an SEM picture of the sample obtained by the process of the present invention after a treatment of 3 weeks;

(5) FIG. 4 shows an SEM picture of the comparative sample after storing a room temperature for 2 weeks;

(6) FIG. 5 shows an SEM picture of the comparative sample after storing a room temperature for 12 weeks; and

(7) FIG. 6 shows an SEM picture of the comparative sample after storing a room temperature for 26 weeks.

(8) In all figures, a scale corresponding to 200 nm is given at the bottom left corner of the respective picture.

(9) As in particular shown in FIG. 2, the desired nanostructures are according to the present invention obtained within a relatively short time frame of only about 2 weeks, whereas for the comparative sample stored at room temperature an acceptable nanostructure formation (i.e. a formation of nanostructures of the desired size, shape and number) has only been observed after 26 weeks.

(10) XPS analysis of the samples according to the present invention revealed a clear increase of the Ti4+ signal (and a decrease of the signal of metallic Ti) in the Ti spectrum, indicating that at least a portion of the nanostructures formed comprise or essentially consist of titanium oxide.

(11) 2. Studies on Impact of Surface Treatment on Protein Adsorption, Blood Coagulation and Osteogenic Differentiation of Human Blood Cells (HBC)

(12) 2.1. Experimental Procedure

(13) 2.1.1. Protein Adsorption

(14) Protein adsorption studies were performed on comparative samples 2.1 as well as on samples 2.2 and 2.3 according to the present invention, the process for obtaining the samples being as follows: Sample 2.1: Roxolid® discs were sandblasted (corundum) with large grits (particle size 250 to 500 μm), then acid-etched in a boiling mixture of HCl and H.sub.2SO.sub.4 followed by cleaning in nitric acid and rinsing in deionized water; finally, the discs were air dried and packed in Al foil. Sample 2.2: The same sandblasting and etching procedure as for sample 2.1 was applied, followed by placing the sample under nitrogen cover gas to prevent exposure to air, by rinsing in 0.9% NaCl solution and by forming nanostructures in a 0.9% NaCl solution at pH 4 to 6. Sample 2.3: The same procedure as for sample 2.2 was applied followed by rinsing with ultrapure water using an ultrasonic bath after several months of storage and by drying the rinsed sample in a stream of N.sub.2 and packing in Al foil like sample 2.1.

(15) Specifically, protein adsorption studies were performed with protein solutions of bovine fibronectin-HiLyte488 (3 μg mL.sup.−1) (Lubio Science) and human fibrinogen Alexa Fluor® 488 conjugate (7 μg mL.sup.−1) (Thermo Fisher Scientific), both dissolved in PBS. For this, surfaces were placed into a 94 well-plate and incubated in 100 μL of protein solution for 15 min at room temperature. Subsequently, surfaces were washed 3× with PBS and analysed with a fluorescence scanner (LS Reloaded™, Tecan Trading AG, Switzerland) using the same settings, i.e. voltage, pinhole, and focal plane, for all samples per condition. Fluorescence intensities (FI) were determined from the images, using the inner 80% surface area of the samples in order to avoid measurement of artefacts at the border of the samples.

(16) 2.1.2. In Vitro Studies

(17) For the in vitro studies, human whole blood was obtained from healthy volunteers by standard venipuncture technique. The blood was partially heparinized directly upon withdrawal into a 9 ml S-Monovette tube with 3 IU ml.sup.−1 sodium heparin (final concentration 0.5 IU heparin/ml blood) and used for the experiments within 1 h after withdrawal.

(18) Samples were placed into a custom-made sample holder made of polytetrafluorethylen (PTFE, Teflon®), which accommodates up to 6 samples. Freshly withdrawn blood (2.8 ml) was added onto the samples. To prevent contact with air the sample holder was closed with a PTFE lid and sealed with parafilm before incubation on a tumbling shaker at 10 rpm at room temperature.

(19) The incubation time was determined for each experiment individually. For this, whole blood was spiked with fluorophore (Alexa 488)-labeled fibrinogen, which allows for live monitoring of the blood coagulation on the samples using fluorescence microscopy. As reference surface, a Roxolid® disc with an SLActive® surface (corresponding to samples 2.2 and 2.3 prior to the nanostructure formation treatment according to the present invention) was used and two time points were chosen representing the beginning of coagulation (t1) and clearly visible layers of fibrin network (t2).

(20) After the period of incubation, blood was removed and the samples were washed 3 times by adding pre-warmed PBS and incubation at 10 rpm for 1 min per washing step. Thereafter, samples were transferred into a 96 well plate.

(21) The samples were analysed using CLSM (confocal laser scanning microscopy) analysis. To this end, the samples were blocked by incubation for 30 min in PBS with 5% goat serum and 1% FCS and platelets were stained with Alexa546-labeled phalloidine for 1 hour at room temperature. The platelets and the fibrin network (visible due to spiking of the blood with Alexa488-labeled fibrinogen) were imaged with a CLSM (10×, 40× magnification). Only one time point (t.sub.2) was imaged. On samples showing complete surface-coverage with fibrin, the thickness of the fibrin network was measured from CLSM z-stack images.

(22) In order to analyse the impact of the surfaces on their osseintegration potential, HBCs were cultivated on top of the whole blood pre-incubated surfaces in osteogenic differentiation media. As reference HBCs were cultured on tissue culture plastic either in proliferation or osteogenic differentiation medium. Mineralisation after 28 days of culture was analysed by measuring the Ca.sup.2+ content in relation to the cell number on the respective surface. For this, HBC proliferation after the period of culture was determined by alamar blue (AB) assay (readout: fluorescence at 530 and 635 nm). AB is reduced by living cells, thereby increasing its own fluorescence. Cell numbers were calculated by interpolating fluorescence readings from a 6-point standard curve (measured from known HBCs numbers after 1 day in culture).

(23) Afterwards, the Ca.sup.2+ content (Quanti Chrom™ Calcium Assay) on the same samples was assessed. For this, all samples were washed twice with pre-warmed PBS without glucose and lysed (upside down) in 100 μl 1 M HCl for 3 h at 37° C. under constant agitation. Subsequently, 10 μl of each supernatant was transferred to a 96 well-plate and 90 μl of working reagent (equal volumes of solution AB; Quanti Chrom™) containing a phenolsulphonephthalien dye, was added. The dye forms a stable blue coloured complex with free calcium. After 3 min, the absorbance of the solution was measured at 595 nm. Samples were analysed in triplicates. Calcium concentrations were calculated by means of a standard curve.

(24) 2.2. Results

(25) 2.2.1. Protein Adsorption

(26) Protein adsorption of fibrinogen and fibronectin on top of the surface of samples 2.1, 2.2 and 2.3 was quantitively analysed using fluorophore labelled proteins and a fluorescence scanner. It was found that both protein interact similarly with different structures, the quantitative assessment of protein concentrations showing higher values for sample 2.2 than for sample 2.3 and higher values for sample 2.3 than for sample 2.1.

(27) Thus, both the hydrophilicity as well as the presence of nanostructures obtainable by the process of the present invention play a role in the adsorption of both fibrinogen and fibronectin.

(28) 2.2.2. Assessment of Fibrin Network and Fibrin Thickness on Surfaces Incubated with Human Whole Blood Via CLSM

(29) The effect of the surfaces on blood coagulation was assessed via CLSM analysing fibrin (fluorophore labelled) and platelets (stained with phalloidin-conjugate).

(30) A qualitative analysis of microscopy images revealed a better coverage for sample 2.2 than for sample 2.3 and a better coverage for sample 2.3 than for comparative sample 2.1

(31) In order to analyse the fibrin coverage in detail, the thickness of the fibrin network was determined in CLSM z-stack images, whereby only surfaces showing a homogenous fibrin network were analysed.

(32) The result of the analysis is given in Table 1, confirming the trend of the qualitative analysis:

(33) TABLE-US-00001 TABLE 1 Mean fibrin Number of thickness on Standard evaluable Sample surface [μm] deviation [μm] experiments 2.1 10.1 0.8 3 2.2 12.8 2.6 2 2.3 11.6 2.8 2
2.2.3. Quantification of HBC Mineralization

(34) The effect of different surfaces on HBC mineralization was assessed by measuring the acellular and cellular Ca.sup.2+ concentration after 28 days of culture in differentiation medium applying the Quanti Chrom™ Calcium Assay kit. Mineralisation was normalized to cell numbers on the respective surfaces obtained from measurements with alamarBlue® cell viability reagent.

(35) Analysing the mineralization of the HBCs after 28 days of culture showed large variations in the observed Ca.sup.2+ concentrations for the different HBC donors. However, the trend observed between the materials was not affected by the donor variations and was similar in all experiments.

(36) To summarize, a higher trend for HBC mineralization was observed for sample 2.2 compared to sample 2.3, and a higher trend was observed for sample 2.2 compared to comparative sample 2.1. Specifically, if compared to comparative sample 2.1, both the surfaces of samples 2.2 and 2.3 according to the present invention have higher levels of mineralization, the effect being statistically significant.